Vancomycin kills Gram-positive enterococci and staphylococci by interfering with cell-wall biosynthesis. We have shown that in wild-type Staphylococcus aureus, vancomycin binds to the stem termini of cell-wall precursors and inhibits transglycosylation (glycan chain extension). Recently, vancomycin-resistant enterococci and staphylococci have emerged. These bacteria have altered peptidoglycan structures and reduced vancomycin binding. We have used solid-state NMR to characterize the cell-wall complexes of five fluorinated glycopeptides with improved potency against vancomycin-resistant enterococci. We obtained two of the drugs from Eli Lilly Company, two from the Cause Institute of New Antibiotics (Russia), and one we synthesized ourselves. We have correlated structure and activity for these drugs for the first time and have formulated their mode of action. In the next grant period, we plan to prove or disprove the general hypothesis, that these drugs kill vancomycin-resistant enterococci and staphylococci by interfering with template recognition during peptidoglycan biosynthesis. We believe that new peptidoglycan strands must be pre-ordered to fit into a tightly cross-linked three-dimensional network, and that this is the reason that an existing nearest-neighbor strand is used as a template for the synthesis of a new strand. We will test these notions using solid-state NMR detection of drug-complex formation and biosynthesis in whole cells of a variety of vancomycin-susceptible and vancomycin-resistant enterococci and staphylococci, organisms which we have acquired from our colleague- collaborators, P. Courvalin (France) and H. Labischinski (Germany). We will use highly selective stable-isotope labeling protocols with detection by new, specially designed solid-state NMR experiments. None of the glycopeptides with improved potency against vancomycin-resistant enterococci and staphylococci that we have examined are approved for clinical use in the United States because of deleterious side effects. We are hopeful that the insights into the mode(s) of action of these glycopeptides that will result from the work proposed in this application will stimulate the search for new and potent antibiotics with tolerable side effects.